All experiments of co-digestion were conducted in 120 mL serum bottles in triplicate. To carry out the digestion, the microorganisms were provided with 50 mL “degassed” inoculum and optimal environmental conditions were assured by 10 mL anaerobic basic medium addition. The substrates were sewage sludge and microalgae, separately or mixed. Blank assays were prepared without substrate addition, and the biogas production was subtracted from the reactors fed with the substrates. The specific methanogenic activity for inoculum was determined with an initial concentration of 1 g/L acetic SB-505124 in the reactor.
Sludge and microalgae were added according to the experimental design. The total substrate amount was decided at 0.12 g volatile solids (VS) equivalent to 2–3 gCOD/L, which generates a measurable but not excessive biogas volume. The reactor feed was based on 100% sludge VS for sewage sludge digestion (denoted as Sludge in the figures), and subsequently 25%, 50%, 75% and 100% of the sludge VS were replaced with microalgae VS respectively (denoted as 25%, 50%, 75% and 100% in the figures). Deionised water was added to a final volume of 80 mL and the reactors were closed with a septum and an aluminium crimp. Finally, the reactors were purged with nitrogen to assure anaerobic conditions and placed into an oven.